Throughout this application various publications are referenced, many in parenthesis. Full citations for each of these publications are provided at the end of the Detailed Description. The disclosures of each of these publications in their entireties are hereby incorporated by reference in this application.
Most infectious disease agents gain entrance to the host through a mucosal surface, therefore the first line of defense is the mucosal immune system. In fact, protection against many microorganisms better correlates with local than systemic immune responses (Galan et al. 1986; Galan and Timaney 1985). The use of non-replicating antigens to stimulate mucosal immune responses has been hampered by the lack of adjuvants that effectively induce secretory immunity. Live, replicating antigens are known to better stimulate mucosal immunity partly because they tend to persist longer (Ganguly and Waldman 1980). Avirulent strains of Salmonella typhimurium endowed with the ability to express cloned genes from other pathogens have been used to stimulate a generalized mucosal immune response against the recombinant virulence antigens (Doggett and Curtiss 1992; Curtiss et al. 1988; Curtiss et al. 1990; Galan et al. 1988). This approach is based on the fact that S. typhimurium invades and proliferates in the gut-associated lymphoid tissue (GALT) (Carter and Collins 1974) and that antigens delivered into the GALT lead to an immune response at other mucosal sites (Cebra et al. 1976).
After oral ingestion, Salmonella typhimurium penetrates the cells of the intestinal epithelium (Takeuchi 1967). Once internalized, Salmonella are translocated through the epithelial cells to the lamina propria where they are later taken up by macrophages. During the translocation process, Salmonella transit inside endocytic vesicles where they undergo limited replication. This is unlike other invasive pathogens, such as Shigella spp. or Listeria monocytogenes, which escape the endocytic vesicles shortly after internalization and actively replicate in the cell cytosol.
The compartment in the eukaryotic cell in which a bacterium resides is very important when it is being considered as an antigen delivery vehicle, because its location will largely determine whether the antigen will be recognized in association with MHC class I or class II molecules. Antigens presented in the context of class I MHC molecules will predominantly induce cytotoxic T cells (T.sub.ctl), while antigens recognized in association with MHC class II molecules will be more likely to stimulate T helper cells (T.sub.H) (Harding et al. 1988; Chain et al. 1988; Allen 1987). This is of great importance in vaccine design since protection against different infectious agents requires different types of immune responses. Thus in general terms, T.sub.ctl 's play a key role in protection against most viral and some intracellular bacterial pathogens while T.sub.H 's are more important in responses against exogenous antigens that enter the processing cells (expressing class II molecules) by endocytosis (Long and Jacobson 1989; Long 1989; Kaufman 1988).
It has been established that processing of exogenous antigens involves endocytosis, partial degradation within the endocytic vacuole, and binding to class II MHC molecules. Processing of class I-restricted antigens also appears to involve proteolysis and recognition of antigen-derived peptides bound to MHC class I molecules, although this processing is not secondary to endocytosis. Rather, antigens synthesized within host cells (e.g., viral proteins), or antigens derived from intracellular bacteria that have the ability to exit the endocytic vacuole (e.g., Listeria monocytogenes and Shigella spp.), are processed and then preferentially associate with MHC class I molecules (Long and Jacobson 1989; Kaufman 1988).
Even though humoral (in particular mucosal) immune responses are an important part of the protective mechanisms against pathogens, it is clear that for efficient protection, cell-mediated immunity is often essential. This is particularly so when the pathogen in question is a virus or an intracellular bacterium. In many of these cases, class I restricted-immune responses are thought to be crucial for protection. This type of immune response is stimulated by proteins that are newly synthesized (e.g., viral antigens) or that otherwise gain access to the cytosol of the infected cell (e.g., Listeria antigens). S. typhimurium has the ability to invade (enter) mammalian cells. Unlike other facultative intracellular pathogens such as Listeria or Shigella spp., which gain access to the cytosol shortly after entry, Salmonella spp. remain inside the endocytic vesicle throughout their entire intracellular life cycle. Although there are some exceptions to this generalization (Aggarwal et al. 1990; Flynn et al. 1990), it appears that Salmonella is not very efficient at stimulating class I-restricted immune responses, which are known to be crucial for protection against viruses and a variety of intracellular pathogens (Gao et al. 1992; Yang et al. 1990). This has been clearly demonstrated using avirulent Salmonella strains expressing different antigens from influenza virus. In a series of very elegant studies (Brett et al. 1993; Tite et al. 1990a; Tite et al. 1990b), it was shown that mice vaccinated with avirulent strains of Salmonella expressing the influenza virus NP failed to mount a significant class I-restricted T cell response against the NP, although they successfully induced class II-restricted responses. On the contrary, class I-restricted responses against the NP were readily demonstrated in mice infected with the virus. As a consequence of this failure, recombinant Salmonella vaccine strains failed to protect mice against influenza virus challenge since in this model of NP immunization, protection is largely dependent on nucleoprotein-specific class I-restricted CD8.sup.+ cells.
An essential feature of the pathogenesis of Salmonella spp. is their ability to stimulate a variety of host-cell responses (reviewed in Galan and Bliska 1996). These responses are largely dependent on the type of cell engaged by the bacteria. For example, in non-phagocytic cells such as those of the intestinal epithelium, Salmonella spp. induce profound cytoskeletal rearrangements, membrane ruffling and macropinocytosis which ultimately result in bacteria internalization. In macrophages, on the other hand, Salmonella spp. induce programmed cell death (Chen et al. 1996). Essential for the stimulation of these responses is the function of a specialized protein secretion system encoded at centisome 63 of the bacterial chromosome (reviewed in Galan 1996). This protein secretion system, termed type III, directs the export of a number of proteins, some of them with presumed effector function. Characteristic features of this protein secretion system, which has also been identified in several other animal and plant pathogenic bacteria, include: 1) the absence in the secreted proteins of a typical, cleavable, sec-dependent, signal sequence; 2) the requirement of several accessory proteins for the export process; 3) the export of the target proteins through both the inner and outer membranes; and 4) the requirement of activating extracellular signals for its full function (reviewed in Galan 1996). Studies of pathogenic Yersinia spp. have established that a similar type III secretion apparatus directs the translocation into the host cells of a number of putative effector proteins such as the bacterial outer proteins YopE, YOpH, YopM and YpkA (Rosqvist et al. 1994; Sory and Cornelis 1994; Persson et al. 1995; Sory et al. 1995; Hakansson et al. 1996). Such translocation is thought to occur in a polarized manner in which proteins are transferred directly from the bacteria to the host cells without secretion into the infection medium. A notion has therefore emerged that protein translocation into host cells is perhaps the main function of this type of protein secretion system. This hypothesis is further supported by the observation that type III protein secretion systems have always been identified as essential determinants involved in intimate interactions of bacterial pathogens with their hosts (reviewed in Galan and Bliska 1996).
Several Salmonella proteins that are exported through this pathway have been identified although it is not known which, if any, of these proteins is translocated into host cells.
A need exists for new methods for stimulating class I-restricted immune responses.